Invasive Chinese Pond Mussel Sinanodonta Woodiana Threatens Native Mussel Reproduction by Inducing Cross‐Resistance of Host Fish

Received: 13 June 2016 Revised: 15 November 2016 Accepted: 8 January 2017 DOI 10.1002/aqc.2759

RESEARCH ARTICLE

Invasive Chinese pond mussel Sinanodonta woodiana threatens native mussel reproduction by inducing cross‐resistance of host fish

1 Department of Zoology and Fisheries, Czech University of Life Sciences Prague, Prague, Abstract Czech Republic 1. The effects of invasive alien species (IAS) on host–affiliate relationships are often subtle and 2 The Key Lab of Aquatic Biodiversity and remain unnoticed or insufficiently quantified. The global decline of freshwater unionid mussel Conservation, Institute of Hydrobiology, species has been attributed to many causes, but little is known about the interactions of IAS, Chinese Academy of Sciences, Wuhan, China with their complex life cycle, which includes an obligatory parasitic stage (the glochidium) that 3 Institute of Vertebrate Biology, Academy of develops on fishes. Sciences of the Czech Republic, Brno, Czech Republic 2. The capacity of a European freshwater mussel, Anodonta anatina, to develop on its widespread 4 CIBIO/InBIO ‐ Research Center in fish host, Squalius cephalus was tested experimentally, after previous infestations by the IAS, Biodiversity and Genetic Resources, University Sinanodonta (Anodonta) woodiana. The initial attachment of glochidia, the length of the parasitic of Porto, Vairão, Portugal period, and the metamorphosis success rate of A. anatina glochidia were compared among 5 CIIMAR/CIMAR ‐ Interdisciplinary Centre of Marine and Environmental Research, treatments of different priming infestation intensities. University of Porto, Matosinhos, Portugal 3. The metamorphosis success rate of the native A. anatina glochidia was strongly reduced 6 – CBMA Centre of Molecular and (Wilcoxon Signed‐Rank Test, P < 0.001) and declined by 42.1 and 45.4% on fish hosts that Environmental Biology, Department of Biology, University of Minho, Braga, Portugal were previously exposed to S. woodiana by single and multiple priming infestations, respec- ‐ Correspondence tively, in comparison with the control group. Such cross resistance is expected to decrease sig- Karel Douda, Czech University of Life Sciences nificantly the quality of the host resources available to native mussels. Prague, Department of Zoology and Fisheries, 4. This study provides the first evidence of the host‐mediated adverse impact of invasive Kamýcká 129, Prague, CZ 165 00, Czech Republic. S. woodiana on native mussel species. These results also highlight the importance of potential Email: [email protected] competition for hosts between threatened groups of affiliate species and their invasive coun- Funding information terparts, which should be reflected in conservation strategies. Czech Science Foundation, Grant/Award Number: 13‐05872S

KEYWORDS

adaptive immunity, Anodonta anatina, competition, freshwater, glochidia, host–parasite relationships, invasive alien species

1 | INTRODUCTION may impair several ecosystem functions because these species have high filtration rates, providing an important link between the water The abundance and diversity of freshwater mussels are severely column and the benthic zone, and also playing an important role in decreasing worldwide, with nearly half of the species currently nutrient cycling, substrate stability, bioturbation, and controlling threatened. This situation may have profound consequences at sev- the levels of suspended solids (Bauer & Wachtler, 2001; Bogan, eral ecological levels, from individuals to ecosystems (Bogan, 1993; 1993; Lopes‐Lima et al., 2017; Vaughn & Hakenkamp, 2001; Burlakova et al., 2011; Lopes‐Lima et al., 2014; Walker, Jones, & Williams, Warren, Cummings, Harris, & Neves, 1993). In addition, Klunzinger, 2014). For example, the decline of freshwater mussels freshwater mussels are usually considered suitable bioindicators of

Aquatic Conserv: Mar Freshw Ecosyst. 2017;1–9.wileyonlinelibrary.com/journal/aqc Copyright © 2017 John Wiley & Sons, Ltd. 1 2 DONROVICH ET AL. overall ecosystem health and are also thought to be keystone spe- suggested that particular mussel species can compete for host cies, as they increase species richness and overall biodiversity resources by inducing cross‐resistance of host fish (Dodd, Barnhart, when present in an ecosystem (Aldridge, Fayle, & Jackson, 2007; Rogers‐Lowery, Fobian, & Dimock, 2005; Strayer, 2008). Competi- Spooner et al., 2013). tion for host fish is most likely a common and natural mechanism Although the reasons for freshwater mussel decline are numer- in freshwater mussel assemblages (Strayer, 2008). However, it could ous and idiosyncratic, one of the causes of most concern is related have detrimental consequences for native mussel species when to the introduction (deliberate or accidental) of invasive alien species induced by IAS. (IAS). The number of freshwater IAS has been increasing rapidly in There is only limited knowledge on the specific impacts that both tropical and temperate latitudes and rigorous studies of the invasive mussels have on native mussels with regard to competition impacts of IAS are often lacking (Tricarico, Junqueira, & Dudgeon, for host fish. In this context, one of the most potentially hazardous 2016). It is supposed that introductions of other freshwater bivalves species is Sinanodonta (Anodonta) woodiana (Lea 1834), commonly are particularly problematic for native freshwater mussels (Baker & known as the Chinese pond mussel. Sinanodonta woodiana is native Levinton, 2003; Ricciardi, Neves, & Rasmussen, 1998; Sousa, Novais, to two river basins in China, the Yangtze and Amur rivers, where it Costa, & Strayer, 2014; Sousa, Pilotto, & Aldridge, 2011). The intro- was primarily located before its expansion to different parts of the duction of bivalve species can lead to competition with native spe- world, including Southeast Asia, Europe, North America, and the cies for space, nutrients, food, and possibly also for host fish, the Caribbean (Kraszewski, 2007; Watters, 1997; Zieritz et al., 2016). latter of which are necessary for completion of the freshwater mus- This species is spreading quickly throughout European waters and sel life cycle (Arey, 1932; Barnhart, Haag, & Roston, 2008; Kat, has reached high densities in many rivers and standing water bodies, 1984; Novais, Dias, & Sousa, 2016; Sousa et al., 2014). such as the Cris/Koros River basin in Romania and Hungary Unionid freshwater mussels produce parasitic larvae (glochidia) (Sarkany‐Kiss, 1997; Sarkany‐Kiss, Sirbu, & Hulea, 2000), Lake that must attach to fish to complete their development into juve- Balaton in Hungary (Benkő‐Kiss, Ferincz, Kováts, & Paulovits, niles (Dillon, 2000). To form a successful host fish and freshwater 2013), the Danube and Tisza rivers in Hungary (Bódis, Tóth, & mussel relationship, three conditions are required: initial contact Sousa, 2014, 2016), lowland rivers in Serbia (Paunovic, Csányi, Simic, between glochidia and the host fish, physiological suitability of Stojanovic, & Cakic, 2006) and the Czech Republic (Douda, Vrtílek, the host for attachment and glochidia development, and resistance Slavík, & Reichard, 2012), channels with soft substrate in Italy of the glochidia to the host’s immune responses (Neves, Weaver, (Cappelletti, Cianfanelli, Beltrami, & Ciutti, 2009), inter‐basin water- & Zale, 1985). In each reproductive year, many glochidia are ways in the Iberian peninsula (Pou‐Rovira et al., 2009), and warmer released, so the first factor is dependent on the distinct infestation water bodies in Poland (Kraszewski & Zdanowski, 2007). Reproduction strategies of the mussels (Barnhart et al., 2008), ecosystem condi- in S. woodiana occurs in autumn and the glochidia are released in tions, fish microhabitat preferences, fish behaviour and fish abun- summer in European waters (Douda et al., 2012; Sarkany‐Kiss et al., dance. If the glochidia fail to attach to a host fish, they will 2000) to begin parasitic life on a host for a period that varies accord- eventually sink to the substrate, where opportunities for attach- ing to environmental conditions, mainly water temperature ment are highly unlikely and the glochidia perish (Jansen, Bauer, (Afanasjev, Zdanowski, & Kraszewski, 2001). Sinanodonta woodiana & Zahner‐Meike, 2001). Unsuccessful infestations can take place produce glochidia two or three times per year (Sarkany‐Kiss et al., either when the host fish contains insufficient chemical or nutri- 2000) in high numbers (Wachtler, Dreher‐Mansur, & Richter, 2001) tional requirements for metamorphosis to occur or as a result of and successfully parasitize a wide spectrum of European native fish direct immune system rejection by the host fish (Neves et al., species (Douda et al., 2012). 1985). In these cases, glochidia may fail to become encysted or The aim of this study was to quantify the capability of a native are sloughed off before transformation (Jansen et al., 2001). Alter- European fish to host native mussel glochidia following previous natively, failure to metamorphose may result from the glochidia infestation by the invasive S. woodiana. The null hypothesis tested being abnormally encysted before detachment (Arey, 1932; was that a priming infestation by an invasive unionid mussel has Rogers‐Lowery, Dimock, & Kuhn, 2007). no detectable effect on the subsequent development of native The fish immune system includes innate and adaptive compo- mussel glochidia. Squalius cephalus (Linneaus 1758), European chub, nents (Lieschke & Trede, 2009). Each of these acts as a means of was chosen as the host species because it has the widest distribu- protection against parasites, including glochidia. Innate immunity tional overlap with S. woodiana and Anodonta anatina (Linnaeus involves the general defence mechanisms that are continuously 1785), and high local abundance in most fish assemblages in present in the fish as a response to foreign substances. Adaptive Central Europe (Douda et al., 2012; Kottelat & Freyhof, 2007; immunity arises as a response to a specific antigen, where the Lopes‐Lima et al., 2017). Squalius cephalus were artificially infested strength of the response is greatly amplified by previous contact (primed) with glochidia of S. woodiana at varying intensities. The with that same antigen. Several studies have found that previously host fish were then re‐infested under laboratory conditions with infested host fish contain large amounts of specific antibodies pro- glochidia of the native duck mussel, A. anatina to determine the duced in response to glochidial infestations (Dodd, Barnhart, success rate of juvenile mussel metamorphosis. Developmental suc- Rogers‐Lowery, Fobian, & Dimock, 2006; O’Connell & Neves, cess of glochidia was compared among treatment groups, and the 1999; Rogers‐Lowery et al., 2007). Because adaptive immunity can potential conservation implications for native freshwater mussels inhibit or prevent secondary infestations in host fish, it has been are discussed. DONROVICH ET AL. 3

2 | METHODS them with 2‐phenoxyethanol (0.2 mL L−1; Merck KGaA, Germany). Passive integrated transponders (PITs; Trovan ID100, 0.1 g in air, 2.1 | Study species 12 × 2.1 mm; EID Aalten B.V., Aalten, the Netherlands) were then inserted into the dorsal musculature using a syringe. Parasitic larvae of the invasive S. woodiana were obtained from an established population in its non‐native Central European range. 2.2 | Laboratory infestations Gravid females were collected in the Kyjovka River (Czech Republic, 48°46’42.07″N, 17°0’57.97″E) and were transferred to stock tanks Three different infestation treatments (reported hereafter as ‘naïve’, (six outdoor aerated fiberglass pools with a total water volume of ‘single priming’ and ‘multiple priming’) were used to compare the 5000 L) at the Czech University of Life Sciences Prague on 24 July effects of previous contact of fish with glochidia. The multiple priming 2015. Gravid A. anatina specimens were collected on 6 November treatment was designed to reach the maximum levels of infestation 2015 from a slow‐moving section of the Sázava River (Czech Republic, reported in literature (tens of glochidia per gram of fish body weight) 49°51’23.59″N, 14°42’0.01″E). The collection was completed using and repeated contact with glochidia throughout the season to mimic the same procedure and equipment as for the collection of S. natural conditions. The single priming treatment received an identical woodiana. glochidia load during the first infestation event, but the fish were not Glochidia were obtained from the parent mussels with ripened lar- infested in subsequent inoculations. The naïve group obtained no vae by flushing the marsupia with water using a syringe immediately glochidia, but the fish were handled in the same way as the infested before each experimental infestation. The viability of glochidia was fish during the experiment. verified by quantifying the closing response to sodium chloride in sub- Immediately before the start of the first infestation, fish were ran- samples (ASTM E2455‐06, 2013). The glochidia from between four domly divided into the three treatment groups (60–75 individuals in and six different gravid females with a glochidia viability exceeding each) and their PIT code numbers were recorded. The fish were 90% were pooled and used for inoculation of all fish to standardize infested in an aerated common bath suspension of dechlorinated tap the potential for infection of all study host individuals. The actual con- water (0.5 L per fish) and live glochidia. The water was stirred manually centration of the inoculation bath was assessed by counting the num- before and during the infestation to maintain a homogeneous suspen- ber of viable glochidia in 10 × 10 mL water samples that were removed sion of glochidia in the water. This process was sustained continuously by a syringe from the baths over the course of inoculation. for 15 min to provide sufficient time for the glochidia to attach to host The test host fish species was S. cephalus. This host species has a fish. After the designated infestation time, the fish were transferred to wide geographical distribution, large ecological tolerance and high local another bath of dechlorinated tap water for 30 min to rinse off population densities (Musil, Horký, Slavík, Zbořil, & Horká, 2012), glochidia that had not attached. ensuring the potential for natural incidence of the initial contact Fish from all treatment groups were subsequently pooled, between glochidia of both study mussel species and host individuals returned to the initial holding system, and kept under the above‐ (Blažek & Gelnar, 2006). On the other hand, metamorphosis success described conditions between infestation events, which were per- rate of the native A. anatina glochidia is relatively lower on Squalius formed using the same methods. Fish were classified by their PIT cephalus compared with some other potential host species (Douda, codes and separated into their respective infestation treatment groups 2015; Douda et al., 2014). This host species was selected because it immediately before each of the subsequent infestations. In total, fish is a known host of both S. woodiana and A. anatina and is widely pres- from the multiple priming treatment group were infested three times ent, and because the species probably represents both a stable and in 10‐day intervals after the first infestation, with the last infestation important host resource for A. anatina in central Europe. The fish used taking place 46 days before the experimental infestation by native A. in the experiment were hatchery‐reared juveniles obtained from a local anatina (see details in Figure 1). Mean (Æ SE) glochidia bath densities fish supplier (Vodňany, Czech Republic) and had no previous contact used for the infestations were 5540 Æ 746, 7200 Æ 1592 and with glochidia. Fish individuals of a similar size (1+ year old; mean stan- 7700 Æ 2113 glochidia per litre in the first, second and third consecu- dard length 128 mm, range 101–144 mm; mean mass 27.5 g, range tive priming infestations, respectively. 13.4–41.6 g) were maintained in an aquarium recirculation system Forty‐six days after the last priming infestation (after all S. composed of four connected holding tanks (320 L each) with a 10% woodiana glochidia detached and before the A. anatina infestation), exchange of fresh water every day. Each tank had an aeration tube, the three distinct groups of S. cephalus were identified by their tag and the water was purified using biological filters (Fluval FX6, Hagen) numbers, and 25 individuals were randomly selected from each group and a UV sterilizer (UV07, Resun). to be infested by glochidia (7433 Æ 1614 glochidia per litre) from A. All fish were acclimated for at least 1 month in the laboratory anatina (a single experimental infestation). All fish were infested in a holding system (dechlorinated tap water, 12‐h light cycle). Tempera- common bath suspension to ensure constant conditions during inocu- ture in the system was recorded with a HOBO data logger (Onset, lation. The infestation procedure followed the same protocol used for USA) at 15 min intervals and was 22.9 Æ 1.4°C (mean Æ SD) over S. woodiana. The length and weight of the experimental fish were the course of the experiment. Fish were fed daily ad libitum during recorded at the end of the experiment, and the relative body weight the acclimation period and the experiment, using commercial fish pel- (the condition factor, K) was calculated using the equation: lets for cyprinids (Biomar Group, Denmark). The fish were tagged K = 100 × somatic weight (g) / (standard length [cm])3 to express the 10 days before the start of the experiment, after first anaesthetizing general condition of the fish individuals (Ricker, 1975). 4 DONROVICH ET AL.

2012) during priming infections as 4.6 Æ 2.3 (15.3 Æ 0.5%) and 10.5 Æ 3.7 (10.6 Æ 1.8%) juveniles per gram of fish body weight were recovered in the single priming and multiple priming treatments, respectively.

2.4 | Statistical analysis

Because the data on glochidial parasitization did not meet the criteria for parametric statistics (visual inspection, Shapiro–Wilk test), and in consideration of the sample size tested (n = 75), non‐parametric tests were used throughout the study. The groups were analysed for potential differences using the non‐parametric Kruskal–Wallis test. When the results of the Kruskal–Wallis test were significant, pairwise com- parisons were conducted according to the two‐tailed unpaired Wilcoxon–Mann–Whitney test and were adjusted for multiple com- parisons using the Bonferroni correction. All statistical analyses were FIGURE 1 The timing of priming (S. woodiana) and experimental performed in R version 3.0.3 (R Development Core Team, 2014). (A. anatina) infestations. Numbers of host fish individuals used are stated in parentheses

3 | RESULTS 2.3 | Monitoring of glochidia development on Clear differences were found in hosting capabilities between naïve and experimental fish primed host fish by invasive S. woodiana (Figure 2). The total number of After the experimental glochidial infestation, the S. cephalus individ- initially attached glochidia per gram was slightly lower in primed host uals were transferred into 18‐L tanks. Each of the 75 tanks fish (Kruskal–Wallis test, n = 75, H = 7.9, df = 2, P < 0.05), and the post contained one fish from an unknown group, chosen at random. hoc test showed that the difference arose from the contrast between The tanks were filled with dechlorinated tap water and maintained naïve and multiple priming treatments (Wilcoxon signed‐rank test, at ambient laboratory temperature 19.3 Æ 1.4°C (mean Æ SD). The monitoring process took place daily for 22 days after the infestation. During the experiment, it was determined that all the fish were in adequate health, and there were no mortalities for the duration of the infestation and monitoring processes. The bottom of each aquarium was covered with a net (mesh size 3 mm) to prevent juvenile predation by host fish, and the water was partially exchanged (approximately 80% of the total water volume) and examined for the presence of glochidia and juvenile mussels daily by siphoning the tank. Glochidia and juvenile mussels were collected from siphoned water using filters (mesh size 139 μm) and were identified under a stereomicroscope at 10‐40× magnification and were counted. Glochidia were recorded as living juveniles if foot activity or valve movement was observed. These methods enabled an esti- mate of both the absolute number of glochidia attached to the fish during the course of the experiment and the metamorphosis success rate of the attached glochidia (Dodd et al., 2005). The same procedure was used to monitor glochidial success during the priming infestations with S. woodiana, using seven extra fish individuals from each of the single priming and multiple priming treatments to verify the effectiveness of the priming infections. Monitoring of these control fish indicated that priming infestations were successful, and S. woodiana juveniles were recovered from all priming infestations performed. The total number of S. woodiana glochidia attached to the FIGURE 2 Developmental dynamics of freshwater mussel A. anatina experimental fish during priming infections was 30.5 Æ 11.6 and glochidia on naïve (a), ‘single priming’ (b), and ‘multiple priming’ (c) S. 101.0 Æ 39.4 glochidia per gram of fish body weight in the single cephalus with glochidia of S. woodiana. Bars represent mean (+SD) priming and multiple priming treatments, respectively. The metamor- glochidia or juveniles detached from fish in the respective day after phic success of S. woodiana was relatively low (cf. Douda et al., infestation DONROVICH ET AL. 5

P < 0.05). The number of successfully metamorphosed juveniles per dead glochidia was significantly reduced from 7.34 Æ 1.26 days in gram of fish body weight was significantly lower for both the single naïve to 6.15 Æ 1.36 and 5.20 Æ 0.89 days in single and multiple prim- and multiple priming treatment than for the naïve host treatment ing treatments, respectively (Kruskal–Wallis test, n = 75, H = 28.9, (Wilcoxon signed‐rank test, P < 0.001). Consequently, the metamor- df = 2, P < 0.001) (Figure 3). phosis success rate of A. anatina glochidia was strongly reduced in The comparison of fish somatic parameters measured at the end both treatments with primed fish (Wilcoxon signed‐rank test, of the experiment revealed no statistically significant differences in P < 0.001). Glochidia on naïve host fish had nearly twice the metamor- fish body weight or condition factor among treatments (Kruskal–Wallis phosis success (5.1 Æ 2.0%) in developing into juvenile mussels com- test, all P > 0.05). The mean Æ SD condition factor was 0.78 Æ 0.05 in pared with glochidia infesting hosts from single (metamorphosis primed fish and 0.79 Æ 0.05 in naïve fish. success 2.9 Æ 1.4%) and multiple (metamorphosis success 2.8 Æ 1.7%) priming treatments (mean Æ SD, Figure 3). Hence, mean glochidial metamorphosis success was reduced in comparison with 4 | DISCUSSION naïve host fish by 42.1 and 45.4% for groups previously infested with

S. woodiana once and three times, respectively. Total numbers (mean- This study documents for the first time a non‐specific resistance of SD) of live A. anatina juveniles recovered per individual fish decreased host fish to glochidia, induced by the invasive unionid S. woodiana. Æ Æ from 80 35 individuals on naïve hosts to 42 22 (single priming) and The transformation success rate of A. anatina was significantly reduced Æ 36 26 (multiple priming) individuals (Figure 3). after priming by the invasive S. woodiana compared with naïve hosts. The length of the parasitic period of successfully metamorphosed Such cross‐resistance can be expected to decrease the quality of host – juveniles did not differ among treatments (Kruskal Wallis test, n = 75, resources available to native mussels in natural habitats. H = 2.1, df = 2, P > 0.05). Nevertheless, because of increased detachment rates of unsuccessful glochidia from host fish during the initial days after infestation (Figure 2), the parasitic period (mean Æ SD) of 4.1 | Adaptive immunity

Based on the results of previous studies on native unionids, the observed cross‐resistance can be attributed to the mechanism of acquired immunity (Rogers & Dimock, 2003). Several studies have found that previously infested host fish can develop specific antibodies against native glochidia (Dodd et al., 2006). The response time of antibody formation for resistance varies among species of fish (Rogers‐ Lowery et al., 2007). Furthermore, the production of antibodies increases both with maturity and rising temperatures (O’Connell & Neves, 1999). Acquired immunity in fish against a specific parasite can persist for months or years, or in some cases perhaps even perma- nently (Dodd et al., 2006). At the intra‐specific level, the previous attachment of glochidia of the broken‐rays mussel Lampsilis reeveiana (Lea 1852), on large‐mouth bass Micropterus salmoides (Lacepede 1802) decreased metamorphosis success with each successive infestation from 67.9% to 38.1% to 28.0% over three infestations (Dodd et al., 2006). These findings can be extended to the inter‐specific level for species from different areas of origin, as the metamorphosis success rate of A. anatina significantly declined after just one priming infestation with the glochidia of the invasive S. woodiana. Nevertheless, it should be mentioned that adaptive immunity is not the only possible explanation for the observed decline in host quality after the priming infestation with glochidia. Previous studies have shown that glochidia of freshwater mussels can have various effects on fish health status and behaviour (Crane, Fritts, Mathis, Lisek, & Barnhart, 2011; Horký, Douda, Maciak, Závorka, & Slavík, 2014; Taeubert & Geist, 2013; Thomas, Taylor, & Garcia de Leaniz, 2013) that could possibly influence glochidial success during the next infestation. These effects were also corroborated with S. woodiana in a study FIGURE 3 Differences in the total number of glochidia attached, that found a significant effect of their glochidia on host condition fac- number of developed juveniles, metamorphosis success rate and duration of parasitism (dead glochidia, live juveniles) between tor, plasma ion concentration and enzyme activities during the parasit- treatment groups. Different letters (a, b, c) indicate significant ization period (Douda et al., 2017). However, the experimental differences between groups (Wilcoxon–Mann–Whitney test, P < 0.05) infestation reported here was performed after a relatively long period 6 DONROVICH ET AL. after the priming infestation (46 days), and no negative effects of the and food resources, change some abiotic features in the sediment, or priming infestations on fish condition were detected. spread diseases and parasites (Sousa et al., 2014); however, all these mechanisms remain untested. Further studies are needed to monitor population trends of native 4.2 | Potential impacts of cross‐resistance on native bivalves at sites invaded by S. woodiana, including the study of addi- mussel species tional combinations of fish and mussel species. In particular, the host The experimental results reported here suggest that the presence of species with higher metamorphosis success rate of A. anatina glochidia S. woodiana can have adverse effects on reproduction and the over- should be tested for cross‐resistance effects because it is still possible all status of freshwater mussels in European waters. This IAS lives in that other hosts are not equally affected by a prior infection by S. various aquatic habitats, such as rivers, lakes, streams, ponds, and woodiana. In terms of the assessment of the potential consequences reservoirs. It is most commonly observed in lowland freshwater envi- of cross‐resistance, more data are needed regarding the strength of ronments, such as ponds, oxbow lakes, canals located on floodplains, the effect, in addition to the duration of the acquired immunity induced and rivers that are slowly to moderately moving (0.05 to 0.3 m s−1), by S. woodiana under natural conditions. There are currently no compre- on muddy sediments where other native unionids are commonly hensive data on S. woodiana glochidia abundance on comparably‐ present (Beran, 2008; Kraszewski & Zdanowski, 2007; Sarkany‐Kiss sized fish. Recently, few parasitizing S. woodiana glochidia per gram et al., 2000). This species has been observed living in sympatry with of fish body weight have been observed in the Kyjovka River native mussel species, such as the painter’s mussel Unio pictorum (Czech Republic) and Bao”an Lake (Hubei province, China) (K. Douda, (Linnaeus 1758), the thick‐shelled river mussel Unio crassus personal observation). Fukuhara (1985) found that the mean number (Philipsson, 1788), the swollen river mussel Unio tumidus (Philipsson, of glochidia per host fish (Rhinogobius brunneus, mean body 1788), the depressed river mussel Pseudanodonta complanata length < 30 mm, weight not provided) exceeded 20 during the main (Rossmässler, 1835), A. anatina, and other mollusc species in the reproduction season and peaked at 52.2 glochidia of S. woodiana per silt–clay substrate of lowland areas of the Danube River (Bódis fish in a pond in Osaka Prefecture (Japan). In light of this information, et al., 2011, 2016; Paunovic et al., 2006). The habitat preferences the numbers of glochidia parasitizing fish during experimental infec- of S. woodiana are similar to those of several native freshwater mus- tions in this study can be supposed as environmentally relevant. Never- sels, which can further increase the risk of host competition with theless, the relationship between the intensity of infestation and the native unionid species. The shells of S. woodiana are also comparable strength of cross‐resistance effects should be studied in detail in future. in size (typically larger) with those of native species (Afanasjev et al., Similarly, the results demonstrate that the number of successful 2001; Hliwa et al., 2015; Kraszewski, 2007) and they are tolerant of juveniles was reduced, but the quality of juveniles recovered from a wide range of environmental conditions (Corsi et al., 2007; Douda primed host fish was not studied (viability and energy reserves of et al., 2012), including polluted and low oxygen ecosystems juvenile mussels can vary among different host fish) (Douda, 2015). (Sarkany‐Kiss et al., 2000). They are host‐generalists (Douda et al., 2012), which can give this species a competitive advantage over 4.3 | Conservation implications of cross‐resistance other mussels. More importantly, the brooding season and the release of glochidia from S. woodiana occurs in advance of several native mus- The adverse impact of S. woodiana priming infestations on the devel- sel species, with S. woodiana releasing glochidia in the summer, in mul- opment of native species’ glochidia reported in this study is the first tiple broods per year (Afanasjev et al., 2001; Douda et al., 2012; evidence of a host‐mediated negative impact of this IAS on native Sarkany‐Kiss et al., 2000). Native Anodonta species release glochidia mussel species. Despite intense concern regarding the potential mainly at the end of winter or early spring (from long‐term brooding) adverse impacts of S. woodiana on biodiversity and ecosystem func- (Hinzmann et al., 2013). Thus, glochidia from S. woodiana are able to tioning (Lopes‐Lima et al., 2017; Sousa et al., 2014; Watters, 1997), infest potential juvenile hosts before other anodontine glochidia. in addition to observational data suggesting that a decline in the rela- No data are available on the population dynamics of native mussel tive density of native mussels occurs following S. woodiana invasion, species after the introduction of the invasive S. woodiana. Neverthe- negative effects were only speculative and were not supported by less, adverse effects are likely, and S. woodiana has already been iden- quantitative data. Consequently, documenting this clear impact on tified as the major cause of decline of native A. anatina in Lake Balaton the developmental success of the glochidia of a native species provides in Central Hungary, where its relative abundance has already dropped a solid basis for conservation and natural resource management deci- from 17.8 to 8.6% (Benkő‐Kiss et al., 2013). In Serbia, S. woodiana has sions, and can also serve as a strong argument for the active control already been shown to outnumber native freshwater mussels by a ratio of S. woodiana invasions. of 2:1 (Paunovic et al., 2006). In Italy, it has completely replaced some Until now, A. anatina has received little attention from a conser- native mussel species (especially A. anatina) in ecosystems throughout vation point of view owing to its wide distribution and high abun- the country (Cappelletti et al., 2009), and the same situation has been dance. However, there is a recent pattern of decline in this species observed recently in southern France (Vincent Prié, personal commu- associated with unspecified human influences and the spread of S. nication). It is important to note that in addition to the mechanism woodiana (Lopes‐Lima, 2014). This, combined with the data provided described in this study (host competition), there are also other hypoth- in the present study, advocates for the strict surveillance of A. eses for the observed reductions in native mussel species after the anatina populations and a reassessment of its conservation status arrival of S. woodiana. For example, the species may compete for space in consideration of the evidence regarding this new threat. DONROVICH ET AL. 7

Because host availability and compatibility is a critical factor in the Benkő‐Kiss, Á., Ferincz, Á., Kováts, N., & Paulovits, G. (2013). Spread and management of populations of endangered unionids (Douda et al., distribution pattern of Sinanodonta woodiana in Lake Balaton. Knowl- edge and Management of Aquatic Ecosystems, 408, 09. 2014; Haag & Stoeckel, 2015; Schwalb, Cottenie, Poos, & Ackerman, Beran, L. (2008). Expansion of Sinanodonta woodiana (Lea, 1834) (Bivalvia: 2011), the comprehensive understanding of fish immunology is impor- Unionidae) in the Czech Republic. Aquatic Invasions, 3,91–94. tant for conservation efforts. Information regarding the immunological Blažek, R., & Gelnar, M. (2006). Temporal and spatial distribution of capabilities of host fish could benefit endangered or threatened glochidial larval stages of European unionid mussels (Mollusca: unionid species (O’Connell & Neves, 1999). Specifically, full awareness Unionidae) on host fishes. Folia Parasitologica, 53,98–106. of the duration, frequency, and mechanisms of adaptive immunity Bódis, E., Nosek, J., Oertel, N., Tóth, B., Hornung, E., & Sousa, R. could aid in the propagation of endangered freshwater mussel species (2011). Spatial distribution of bivalves in relation to environmental worldwide (Dodd et al., 2006). Understanding these timeframes can conditions (middle Danube catchment, Hungary). Community Ecology, 12, 210–219. lead to efficient infestations of host fish in breeding programmes and Bódis, E., Tóth, B., & Sousa, R. (2014). Massive mortality of invasive thereby contribute to artificial reproduction. More importantly, under- bivalves as a potential resource subsidy for the adjacent terrestrial food standing the mechanisms influencing host compatibility (including the web. Hydrobiologia, 735, 253–262. adverse effects of IAS) is essential for ensuring adequate host Bódis, E., Tóth, B., & Sousa, R. (2016). Freshwater mollusc assemblages resources for endangered mussel species in natural habitats. and habitat associations in the Danube River drainage, Hungary. The results of this study can contribute to research efforts on the Aquatic Conservation: Marine and Freshwater Ecosystems, 332, 319–332. potential effects of an IAS on native mussel species. These findings can ‐ help identify the role of fish immunology on secondary glochidial infes- Bogan, A. E. (1993). Fresh water bivalve extinctions (Mollusca, Unionoida) – a search for causes. American Zoologist, 33, 599–609. tations and determine the propensity of native fish species to host Burlakova, L. E., Karatayev, A. Y., Karatayev, V., May, M. E., Bennett, D. L., & multiple mussel species in succession. Multiple glochidial releases are Cook, M. J. (2011). Biogeography and conservation of freshwater mus- common for S. woodiana under natural conditions, and the results indi- sels (Bivalvia: Unionidae) in Texas: Patterns of diversity and threats. cate that the likelihood of native juvenile freshwater mussels develop- Diversity and Distributions, 17, 393–407. ing in ecosystems containing this invasive species can be significantly Cappelletti, C., Cianfanelli, S., Beltrami, M. E., & Ciutti, F. (2009). reduced. These results can potentially serve to accelerate conservation Sinanodonta woodiana (Lea, 1834) (Bivalvia: Unionidae): A new non‐indigenous species in Lake Garda (Italy). Aquatic Invasions, 4, efforts for native freshwater mussels in Europe and to help protect 685–688. them against the impacts of IAS such as S. woodiana. Corsi, I., Pastore, A. M., Lodde, A., Palmerini, E., Castagnolo, L., & Focardi, S. (2007). Potential role of cholinesterases in the invasive capacity of ACKNOWLEDGEMENTS the freshwater bivalve, Anodonta woodiana (Bivalvia: Unionacea): A comparative study with the indigenous species of the genus, All experimental procedures were approved by the Expert Committee Anodonta sp. Comparative Biochemistry and Physiology, Part C, 145, for Animal Conservation (research protocol no. MSMT‐31220/ 413–419. ‐ 2014 6). The research was funded by the Czech Science Foundation Crane, A. L., Fritts, A. K., Mathis, A., Lisek, J. C., & Barnhart, M. C. (2011). Do (13‐05872S). gill parasites influence the foraging and antipredator behaviour of rain- bow darters, Etheostoma caeruleum? Animal Behaviour, 82, 817–823. REFERENCES Dillon, R. T. (2000). The ecology of freshwater molluscs. Port Chester, NY, USA: Cambridge University Press. Afanasjev, S. A., Zdanowski, B., & Kraszewski, A. (2001). Growth and population structure of the mussel Anodonta woodiana (Lea, 1834) Dodd, B. J., Barnhart, M. C., Rogers‐Lowery, C. L., Fobian, T. B., & (Bivalvia, Unionidae) in the heated Konin lakes system. Archives of Polish Dimock, R. V. (2005). Cross‐resistance of largemouth bass to glochidia Fisheries, 9, 123–131. of unionid mussels. Journal of Parasitology, 91, 1064–1072. Dodd, B. J., Barnhart, M. C., Rogers‐Lowery, C. L., Fobian, T. B., & Aldridge, D. C., Fayle, T. M., & Jackson, N. (2007). Freshwater mussel abun- Dimock, R. V. (2006). Persistence of host response against glochidia dance predicts biodiversity in UK lowland rivers. Aquatic Conservation: larvae in Micropterus salmoides. Fish and Shellfish Immunology, 21, Marine and Freshwater Ecosystems, 17, 554–564. 473–484. Arey, L. B. (1932). The formation and structure of the glochidial cyst. Biolog- Douda, K. (2015). Host‐dependent vitality of juvenile freshwater mussels: – ical Bulletin, 62, 212 221. Implications for breeding programs and host evaluation. Aquaculture, 445,5–10. ASTM E2455‐06. (2013). Standard Guide for Conducting Laboratory Toxicity Tests with Freshwater Mussels. West Conshohocken, PA, USA: ASTM Douda, K., Sell, J., Kubíková‐Peláková, L., Horký, P., Kaczmarczyk, A., & International. Mioduchowska, M. (2014). Host compatibility as a critical factor in management unit recognition: Population‐level differences in mussel– Baker, S. M., & Levinton, J. S. (2003). Selective feeding by three native fish relationships. Journal of Applied Ecology, 51, 1085–1095. North American freshwater mussels implies food competition with š ř zebra mussels. Hydrobiologia, 505,97–105. Douda, K., Velí ek, J., Kolá ová, J., Rylková, K., Slavík, O., Horký, P., & Langrová, I. (2017). Direct impact of invasive bivalve (Sinanodonta Barnhart, M. C., Haag, W. R., & Roston, W. N. (2008). Adaptations to host woodiana) parasitism on freshwater fish physiology: Evidence and impli- infection and larval parasitism in Unionoida. Journal of the North cations. Biological Invasions, 19, 989–999. – American Benthological Society, 27, 370 394. Douda, K., Vrtílek, M., Slavík, O., & Reichard, M. (2012). The role of host specificity in explaining the invasion success of the freshwater mussel Bauer, G., & Wachtler, K. (2001). Environmental relationships of naiads: Anodonta woodiana in Europe. Biological Invasions, 14, 127–137. Threats, impact on the ecosystem, indicator function. In G. Bauer, & K. Wachtler (Eds.), Ecology and evolution of the freshwater mussels Fukuhara, S. (1985). Glochidium parasitic period, host‐fish and parasitic Unionoida. (pp. 311–316). Heidelberg, Germany: Springer. site of Anodonta woodiana in small pond. Venus, 45,43–52. 8 DONROVICH ET AL.

Haag, W. R., & Stoeckel, J. A. (2015). The role of host abundance in regulat- R Development Core Team. (2014). R: A language and environment for ing populations of freshwater mussels with parasitic larvae. Oecologia, statistical computing. Vienna, Austria: R Foundation for Statistical 178, 1159–1168. Computing. Hinzmann, M., Lopes‐Lima, M., Teixeira, A., Varandas, S., Sousa, R., Lopes, Ricciardi, A., Neves, R. J., & Rasmussen, J. B. (1998). Impending extinctions A., … Machado, J. (2013). Reproductive cycle and strategy of of North American freshwater mussels (Unionoida) following the zebra Anodonta anatina (L., 1758): Notes on hermaphroditism. Journal of mussel (Dreissena polymorpha) invasion. Journal of Animal Ecology, 67, Experimental Zoology Part A: Ecological Genetics and Physiology, 319, 613–619. – 378 390. Ricker, W. E. (1975). Computation and interpretation of biological statistics Hliwa, P., Zdanowski, B., Dietrich, G. J., Andronowska, A., Krol, J., & of fish populations. Bulletin of the Fisheries Research Board of Canada, Andrzej, C. (2015). Temporal changes in gametogenesis of the invasive 191,1–382. Chinese pond mussel Sinanodonta woodiana (Lea, 1834) (Bivalvia: Rogers, C. L., & Dimock, R. V. (2003). Acquired resistance of bluegill sunfish Unionidae) from the Konin Lakes system (Central Poland). Folia Lepomis macrochirus to glochidia larvae of the freshwater mussel – Biologica, 63,63 67. Utterbackia imbecillis (Bivalvia: Unionidae) after multiple infections. Horký, P., Douda, K., Maciak, M., Závorka, L., & Slavík, O. (2014). Par- Journal of Parasitology, 89,51–56. ‐ asite induced alterations of host behaviour in a riverine fish: The Rogers‐Lowery, C. L., Dimock, R. V., & Kuhn, R. E. (2007). Antibody effects of glochidia on host dispersal. Freshwater Biology, 59, response of bluegill sunfish during development of acquired resistance – 1452 1461. against the larvae of the freshwater mussel Utterbackia imbecillis. Devel- Jansen, W., Bauer, G., & Zahner‐Meike, E. (2001). Glochidial mortality in opmental and Comparative Immunology, 31, 143–155. freshwater mussels. In G. Bauer, & K. Wachtler (Eds.), Ecology and Sarkany‐Kiss, A. (1997). The present‐day situation of the Unionidae – evolution of the freshwater mussels Unionoida. (pp. 185 212). (Mollusca: Bivalvia) in the Transylvanian tributaries of the Tisa River ‐ Berlin/Heidelberg: Springer Verlag. (Romania). Travaux du Museum National d’Histoire Naturelle ‘Grigore Kat, P. W. (1984). Parasitism and the Unionacea (Bivalvia). Biological Antipa’, 37, 213–224. – Reviews of the Cambridge Philosophical Society, 59, 189 207. Sarkany‐Kiss, A., Sirbu, I., & Hulea, O. (2000). Expansion of the adventive Kottelat, M., & Freyhof, J. (2007). Handbook of European freshwater fishes. species Anodonta woodiana (Lea, 1834) (Mollusca, Bivalvia, Unionoidea) Cornol, Switzerland: Publications Kottelat. in central and eastern Europe. Acta Oecologica, 7,49–57. Kraszewski, A. (2007). The continuing expansion of Sinanodonta woodiana Schwalb, A. N., Cottenie, K., Poos, M. S., & Ackerman, J. D. (2011). Dispersal (Lea, 1834) (Bivalvia: Unionidae) in Poland and Europe. Folia limitation of unionid mussels and implications for their conservation. Malacologica, 15,65–69. Freshwater Biology, 56, 1509–1518. Kraszewski, A., & Zdanowski, B. (2007). Sinanodonta woodiana (Lea , 1834) Sousa, R., Novais, A., Costa, R., & Strayer, D. L. (2014). Invasive bivalves in (Mollusca) – a new mussel species in Poland: Occurrence and habitat fresh waters: Impacts from individuals to ecosystems and possible con- preferences in a heated lake system. Polish Journal of Ecology, 55, trol strategies. Hydrobiologia, 735, 233–251. – 337 356. Sousa, R., Pilotto, F., & Aldridge, D. C. (2011). Fouling of European freshwa- Lieschke, G. J., & Trede, N. S. (2009). Fish immunology. Current Biology, 19, ter bivalves (Unionidae) by the invasive zebra mussel (Dreissena 678–682. polymorpha). Freshwater Biology, 56, 867–876. Lopes‐Lima, M. (2014). Anodonta anatina. The IUCN Red List of Threatened Spooner, D. E., Frost, P. C., Hillebrand, H., Arts, M. T., Puckrin, O., & Species 2014: e.T155667A21400363. Xenopoulos, M. A. (2013). Nutrient loading associated with agriculture ‐ Lopes‐Lima, M., Sousa, R., Geist, J., Aldridge, D. C., Araujo, R., Bergengren, J., land use dampens the importance of consumer mediated niche con- – … Van Damme, D. (2017). Conservation status of freshwater mussels in struction. Ecology Letters, 16, 1115 1125. Europe: State of the art and future challenges. Biological Reviews, 92, Strayer, D. L. (2008). Freshwater mussel ecology: A multifactor approach 572–607. to distribution and abundance. Berkley, CA, USA: University of Lopes‐Lima, M., Teixeira, A., Froufe, E., Lopes, A., Varandas, S., & Sousa, R. California Press. (2014). Biology and conservation of freshwater bivalves: Past, present Taeubert, J. E., & Geist, J. (2013). Critical swimming speed of brown trout and future perspectives. Hydrobiologia, 735,1–13. (Salmo trutta) infested with freshwater pearl mussel (Margaritifera Musil, J., Horký, P., Slavík, O., Zbořil, A., & Horká, P. (2012). The margaritifera) glochidia and implications for artificial breeding of an – response of the young of the year fish to river obstacles: Functional endangered mussel species. Parasitology Research, 112, 1607 1613. and numerical linkages between dams, weirs, fish habitat guilds and Thomas, G. R., Taylor, J., & Garcia de Leaniz, C. (2013). Does the parasitic biotic integrity across large spatial scale. Ecological Indicators, 23, freshwater pearl mussel M. margaritifera harm its host? Hydrobiologia, 634–640. 735, 191–201. Neves, R. J., Weaver, L. R., & Zale, A. V. (1985). An evaluation of host fish Tricarico, E., Junqueira, A. O., & Dudgeon, D. (2016). Alien species in suitability for glochidia of Villosa vanuxemi and V. nebulosa (Pelecypoda: aquatic environments: A selective comparison of coastal and inland Unionidae). American Midland Naturalist, 113,13–19. waters in tropical and temperate latitudes. Aquatic Conservation: Marine – Novais, A., Dias, E., & Sousa, R. (2016). Inter‐ and intraspecific variation of and Freshwater Ecosystems, 26, 872 891. carbon and nitrogen stable isotopes ratios in freshwater bivalves. Vaughn, C. C., & Hakenkamp, C. C. (2001). The functional role of Hydrobiologia, 765, 149–158. burrowing bivalves in freshwater ecosystems. Freshwater Biology, – O’Connell, M. T., & Neves, R. J. (1999). Evidence of immunological 46, 1431 1446. responses by a host fish (Ambloplites rupestris) and two non‐host fishes Wachtler, K., Dreher‐Mansur, M. C., & Richter, T. (2001). Larval types (Cyprinus carpio and Carassius auratus) to glochidia of a freshwater mus- and early postlarval biology in naiads (Unionoida). In G. Bauer, & sel (Villosa iris). Journal of Freshwater Ecology, 14,71–78. K. Wachtler (Eds.), Ecology and evolution of the freshwater mussels – ‐ Paunovic, M., Csányi, B., Simic, V., Stojanovic, B., & Cakic, P. (2006). Distri- Unionoida. (pp. 93 126). Berlin/Heidelberg: Springer Verlag. bution of Anodonta (Sinanodonta) woodiana (Lea, 1834) in inland waters Walker, K. F., Jones, H., & Klunzinger, M. W. (2014). Bivalves in a bottle- of Serbia. Aquatic Invasions, 1, 154–160. neck: Taxonomy, phylogeography and conservation of freshwater – Pou‐Rovira, Q., Araujo, R., Boix, D., Clavero, M., Feo, C., Ordeix, M., & mussels (Bivalvia: Unionoida) in Australasia. Hydrobiologia, 735,61 79. Zamora, L. (2009). Presence of the alien Chinese pond mussel Anodonta Watters, G. T. (1997). A synthesis and review of the expanding range of the woodiana (Lea, 1834) (Bivalvia, Unionidae) in the Iberian Peninsula. Asian freshwater mussel Anodonta woodiana (Lea, 1834) (Bivalvia: Graellsia, 65,67–70. Unionidae). Veliger, 40, 152–156. DONROVICH ET AL. 9

Williams, J. D., Warren, M. L., Cummings, K. S., Harris, J. L., & Neves, R. J. (1993). Conservation status of freshwater mussels of the United States How to cite this article: Donrovich SW, Douda K, and Canada. Fisheries, 18,6–22. Plechingerová V, et al. Invasive Chinese pond mussel Zieritz, A., Lopes‐Lima, M., Bogan, A., Sousa, R., Walton, S., Rahim, K., Sinanodonta woodiana threatens native mussel reproduction … McGowan, S. (2016). Factors driving changes in freshwater mus- by inducing cross‐resistance of host fish. Aquatic Conserv: Mar sel (Bivalvia, Unionida) diversity and distribution in Peninsular – Malaysia. Science of the Total Environment, 571, 1069–1078. Freshw Ecosyst. 2017;00:1 9. https://doi.org/10.1002/aqc.2759